The Integrin Ligand MFG-E8 and Photoreceptor Outer Segment Renewal.

描述(申请人提供)：老化的光感受器外节尖端(OS)的脱落和它们被视网膜色素上皮(RPE)吞噬，每天都在视网膜发生。在啮齿动物和人类中，RPE细胞不能吞噬OS会导致视网膜营养不良。此外，延缓的OS退化可能导致老年性黄斑变性的发生或进展。因此，了解外段更新的分子机制是很重要的。我们已经在完整的视网膜中发现了一条信号通路，这是迅速清除OS所必需的。缺乏α-v-Beta5整合素受体的小鼠缺乏同步吞噬功能，导致与年龄相关的光感受器功能障碍和脂褐素积聚。整合素信号需要整合素受体的配体参与。因此，我们假设整合素配体/S的及时出现或募集有助于刺激视网膜的节律性信号。到目前为止，还没有确定这样的整合素配体。我们的初步数据表明，分泌的整合素配体蛋白MFG-E8可能具有这一功能。MFG-E8介导依赖整合素的凋亡细胞吞噬，这一过程类似于OS吞噬。MFG-E8在体外由RPE表达，参与RPE的吞噬作用。该探索性项目将实现两个具体目标：目标1将在缺乏MFG-E8的新小鼠模型中探索视觉功能、视网膜形态、体内和体外RPE吞噬和吞噬信号。这些实验将确定MFG-E8是否作为视网膜中av|35整合素的必要配体发挥作用。目的2研究MFG-E8在视网膜下间隙的活性是否与avB5整合蛋白的激活、每天的OS脱落和吞噬功能有关。它将进一步探索注定要脱落的光感受器尖端是否暴露磷脂酰丝氨酸来增加MFG-E8结合位点。我们将结合分子生物学、显微镜和生化来检测MFG-E8在小鼠视网膜中的转录、蛋白表达和蛋白定位与外节更新的时间进程的关系。我们将开发新的亲和分析方法来定量在完整视网膜中OS脱落时磷脂酰丝氨酸暴露和MFG-E8结合。这一探索性项目的结果是全面研究光感受器末端和分泌蛋白变化的重要前提，这些变化促进了对长期视网膜功能至关重要的外节及时更新。

DESCRIPTION (provided by applicant): Shedding of aged photoreceptor outer segment tips (OS) and their phagocytosis by the retinal pigment epithelium (RPE) take place daily in the retina. Failure of RPE cells to phagocytose OS causes retinal dystrophy in rodents and humans. Furthermore, delayed OS degradation may contribute to development or progression of age-related macular degeneration. It is thus important to understand the molecular mechanisms of outer segment renewal. We have identified a signaling pathway in intact retina that is required to clear shed OS promptly. Lack of synchronized phagocytosis in mice deficient in alpha-v-beta5 integrin receptors causes age-related photoreceptor dysfunction and lipofuscin accumulation. Integrin signaling requires ligand engagement of integrin receptors. We thus hypothesize that timely appearance or recruitment of integrin ligand/s serves to stimulate rhythmic signaling in the retina. No such integrin ligand has been identified to date. Our preliminary data suggest the secreted integrin ligand protein MFG-E8 as candidate for this function. MFG-E8 mediates integrin-dependent phagocytosis of apoptotic cells, a process similar to OS phagocytosis. MFG-E8 is expressed by the RPE and participates in RPE phagocytosis in vitro. This exploratory project will achieve two specific objectives: Aim 1 will explore visual function, retinal morphology, in vivo and in vitro RPE phagocytosis and phagocytic signaling in a new mouse model that lacks MFG-E8. These experiments will determine if MFG-E8 functions as a necessary ligand for av|35 integrin in the retina. Aim 2 will investigate if MFG-E8 activity in the sub-retinal space correlates with avB5 ihtegrin's activation, daily OS shedding and phagocytosis. It will further explore if photoreceptor tips destined for shedding expose phosphatidylserine to increase MFG-E8 binding sites. We will combine molecular biology, microscopy, and biochemistry to detect MFG-E8 transcription, protein expression, and protein localization in mouse retina in relation to the time course of outer segment renewal. We will develop novel affinity assays to quantify phosphatidylserine exposure and MFG-E8 binding at the time of OS shedding in intact retina. The results of this exploratory project are an important prerequisite for comprehensive studies of changes in photoreceptor tip and secreted proteins that promote the timely outer segment renewal that is essential for long-term retinal function.